Remodeling of secretory lysosomes during education tunes functional potential in NK cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation;however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI (3,5) P-2 synthesis, or genetic silencing of the PI(3,5) P-2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education

The importance of nerve microenvironment for schwannoma development

Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas

Untersuchungen zu den Uhrproteinen PERIOD und TIMELESS aus der Fruchtfliege Drosophila melanogaster

In der vorliegenden Dissertation wurden zwei Projekte bearbeitet, die sich mit den Faktoren period (per ) und timeless (tim) des molekularen Oszillators der inneren Uhr von Drosophila melanogaster, beschäftigen. Beide Gene arbeiten in einer negativen Rückkopplungsschleife zusammen und fungieren als Repressoren ihrer eigenen Transkription. Beide Proteine sind jedoch auch an der Verarbeitung äußerer Einflüsse im circadianen System beteiligt. Das Protein TIM stellt eine Schnittstelle des molekularen Oszillators mit dem Mechanismus der Lichtrezeption dar. Eine Aktivierung des Photorezeptors CHRYPTOCHROME (CRY) durch Licht führt zu einem Abbau des Proteins TIM, wodurch die Synchronisation der inneren Uhr mit dem Zeitgeber Licht bewerkstelligt wird. Bereits vor 10 Jahren wurde anhand von Sequenzvergleichen gezeigt, dass es in der Natur zwei unterschiedliche tim-Allele gibt (s-tim und ls-tim), die theoretisch dazu in der Lage sind, zwei unterschiedlich lange TIM-Proteine zu bilden (Rosato et al., Nucleic Acids Res 25, 1997). Das Allel s-tim ist das ursprüngliche Allel und nur in der Lage das kurze S-TIM (1398 Aminosauren) zu bilden. ls-tim ist in Europa durch eine natürliche Mutation aus s-tim entstanden, kurz nachdem Drosophila aus Afrika eingewandert war. Es hat sich dann in Europa verbreitet, da es eine bessere Anpassung an die ausgeprägten jahreszeitlichen Schwankungen von Temperatur und Tageslänge vor allem in Nordeuropa bietet (Tauber et al., Science 316, 2007). Es wurde vermutet, dass es sowohl S-TIM, als auch das längere L-TIM (1421 Aminosäuren) exprimieren kann. Durch Westernblotanalyse der Kopfextrakte verschiedener Fliegenlinien und Sequenzanalyse deren tim-Lokusse konnte gezeigt werden, dass zwei im Gel unterschiedlich schnell laufende TIM-Banden, auf die zwei tim-Allele zurückzuführen sind. Durch Wiederholung der Analysen mit und ohne Phosphatasebehandlung der Proteinextrakte konnte Phosphorylierung als Ursache für das unterschiedliche Laufverhalten der Proteine ausgeschlossen werden. Damit konnte gezeigt werden, dass das neue ls-tim-Allel tatsächlich ein längeres TIM exprimieren kann, als das ursprüngliche s-tim (Sandrelli et al., Science 316, 2007). Das Protein PER ist, wie z.B. die Mutation perL (Konopka and Benzer, Proc Natl Acad Sci USA 68, 1971) oder Untersuchungen der T/G-Region in PER (Sawyer et al., Science 278, 1997) zeigen, unter Anderem auch maßgeblich an der Temperaturkompensation beteiligt, die dafur sorgt, dass sich die Periodenlänge der inneren Uhr nicht temperaturbedingt ändert. Altere Studien haben gezeigt, dass PER nicht nur einen Heterodimer mit TIM, sondern auch einen PER:PER-Homodimer bilden kann (Zeng et al., Nature 380, 1996). Da die Mutation in perL eine Homodimerisierung in vitro verhindert, wurde gefolgert, dass der PER:PER-Homodimer an der Temperaturkompensation beteiligt sein könnte (Huang et al., Science 267, 1995). Die Aufklärung der 3D-Struktur eines PER-Fragments schließlich ergab ebenfalls einen Homodimer und offenbarte zudem dessen Interaktionspunkte, was eine gezielte Untersuchung des Homodimers durch rekombinante Proteine ermöglichte (Yildiz et al., Molecular Cell 17, 2005). Zur Untersuchung der Homodimerisierung von PER wurden daher in dieser Arbeit transgene Fliegenlinien erzeugt, die getagte PER-Proteine exprimieren. Ein Teil dieser Proteine trägt außerdem Aminosäureaustausche, die anhand der Strukturvorhersage eine Homodimerisierung verhindern. Durch Untersuchung des Lokomotorverhaltens der Transformanten, sowie der Expression und Dimerisierung der rekombinanten PER-Proteine, konnte eine Homodimerisierung klar nachgewiesen werden. Außerdem konnte eine Beteiligung am Mechanismus der Temperaturkompensation ausgeschlossen werden. Diese Untersuchungen geben zudem Grund zur Annahme, dass die Bindung von PER an TIM ein wesentlicher Baustein der Temperaturkompensation ist. Der Vergleich der Aminosäuresequenz von PER mit PER-Proteinen der Maus zeigte, dass es auch im PER von Drosophila wahrscheinlich ein Nuclear Export Signal (NES) gibt (Vielhaber et al., J Biol Chem 276, 2001). per-Transformanten, die in diesem Exportsignal eine Mutation tragen, zeigten einen deutlichen Effekt in Bezug auf die zeitliche Kernlokalisation und auch die Temperaturkompensation der inneren Uhr. Dies ist ein guter Hinweis, dass es sich um ein funktionelles NES handelt und zeigt darüber hinaus, dass die zeitlich korrekte Lokalisation von PER einen weiteren wichtigen Bestandteil des Mechanismus der Temperaturkompensation darstellt

Acoustic Limescale Layer and Temperature Measurement in Ultrasonic Flow Meters

Guided acoustic waves are commonly used in domestic water meters to measure the flow rate. The accuracy of this measurement method is affected by factors such as variations in temperature and limescale deposition inside of the pipe. In this work, a new approach using signals from different sound propagation paths is used to determine these quantities and allow for subsequent compensation. This method evaluates the different propagation times of guided Lamb waves in flow measurement applications. A finite element method-based model is used to identify the calibration curves for the device under test. The simulated dependencies on temperature and layer thickness are validated by experimental data. Finally, a test on simulated data with varying temperatures and limescale depositions proves that this method can be used to separate both effects. Based on these values, a flow measurement correction scheme can be derived that provides an improved resolution of guided acoustic wave-based flow meters

A Phenotypic Screening Approach to Identify Anticancer Compounds Derived from Marine Fungi

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R-2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner

C77G in PTPRC (CD45) is no risk allele for ovarian cancer, but associated with less aggressive disease

The pan lymphocyte marker CD45 exists in various isoforms arising from alternative splicing of the exons 4, 5 and 6. While naïve T cells express CD45RA translated from an mRNA containing exon 4, exons 4–6 are spliced out to encode the shorter CD45R0 in antigen-experienced effector/memory T cells. The SNP C77G (rs17612648) is located in exon 4 and blocks the exon’s differential splicing from the pre-mRNA, enforcing expression of CD45RA. Several studies have linked C77G to autoimmune diseases but lack of validation in other cohorts has left its role elusive. An incidental finding in an ovarian cancer patient cohort from West Norway (Bergen region, n = 312), suggested that the frequency of C77G was higher among ovarian cancer patients than in healthy Norwegians (n = 1,357) (3.0% vs. 1.8% allele frequency). However, this finding could not be validated in a larger patient cohort from South-East Norway (Oslo region, n = 1,198) with 1.2% allele frequency. Hence, C77G is not associated with ovarian cancer in the Norwegian population. However, its frequency was increased in patients with FIGO stage II, endometrioid histology or an age at diagnosis of 60 years or older indicating a possible association with a less aggressive cancer type

Comparing the epigenetic landscape in myonuclei purified with a PCM1 antibody from a fast/glycolytic and a slow/oxidative muscle

Muscle cells have different phenotypes adapted to different usage, and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of the epigenetic landscape by ChIP-Seq in two muscle extremes, the fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where up to 60% of the nuclei can be of a different origin. Since cellular homogeneity is critical in epigenome-wide association studies we developed a new method for purifying skeletal muscle nuclei from whole tissue, based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labelling and a magnetic-assisted sorting approach, we were able to sort out myonuclei with 95% purity in muscles from mice, rats and humans. The sorting eliminated influence from the other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the differences in the functional properties of the two muscles, and revealed distinct regulatory programs involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles were also regulated by different sets of transcription factors; e.g. in soleus, binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SIX1 binding sites were found to be overrepresented. In addition, more novel transcription factors for muscle regulation such as members of the MAF family, ZFX and ZBTB14 were identified